**3.2.2.1 Sonification parameters**

In the experiment, the frequency of applied ultrasounds was 20 kHz and the power input was 1.0 W L-1. The initial experiments were done to find the best irradiation period. The experiments revealed, that continuous low energy ultrasound irradiation during 12, 24 and 36 h did not enhance ethanol productivity by co-immobilized *S. cerevisiae*, moreover the ethanol yield coefficients were lower than those obtained in experiments without ultrasound irradiation. The subsequent experiments were carried out with time intervals with and without ultrasonic irradiation in order to obtain the positive influence of ultrasound on biological activity of *S. cerevisiae*. The results showed that the culture should have been sonicated for 1 min every 6 h. It was similarly to results obtained by Marques et al. (2006). They investigated the effect of ultrasound pulses on enzymatic activity of *S. cerevisiae*. Their results showed that the ultrasound pulse at low frequency (20–25 kHz) for a short sonification period of 1 and 2 min increased cell permeability, and the viability rate of yeasts was over 95%. However, in the 4 min sonification, the rate decreased to 46%.

The use of ultrasounds to stimulate biological activity and ethanol production by *S. cerevisiae*  are reported by Schläfer et al. (2000). After testing several different frequencies and power levels, they carried out the experiments at 25 kHz, 0.3 and 12 W L-1. At an ultrasound intensity of 12 W L-1 there was no recognizable difference in the biological activity of yeasts with and without ultrasound. The authors stated that some pauses are needed between ultrasound exposure to obtain positive effects on biological activity of yeast *S. cerevisiae*. Moreover, an increase in biological activity appeared after irradiation and high activity of ultrasound activated cultures stopped for some hours after irradiation. The authors stated,

Feasibility of Bioenergy Production from

immobilized *S. cerevisiae*

Lactose consumption (%)

Ethanol concentration (g L-1)

Ultrafiltration Whey Permeate Using the UASB Reactors 207

Ultrasonic irradiation Without ultrasonic irradiation

12 24 36

12 24 36

HRT (h)

Fig. 10. Effects of HRT and ultrasound irradiation on the lactose consumption by co-

Fig. 9. Effects of HRT and ultrasound irradiation on the ethanol concentration

HRT (h)

Ultrasonic irradiation Without ultrasonic irradiation

that discontinuous ultrasonic irradiation of *S. cerevisiae* was more beneficial for activating fermentation than the continuous exposure, because only a few steps in intracellular metabolisms are supported by ultrasound and others are not or even inhibited.

Liu et al. (2007) investigated the changes of biological activity of aerobic activated sludge after ultrasonic irradiation. The activity of microorganisms rose sharply after ultrasonic exposure of 0.3 W cm2, 35 kHz for 10 min, and reached a peak level in 8 h after exposure (100% higher than that of the initial level immediately after exposure). Then it dropped rapidly in the next 8 h. In 24 h after ultrasonic irradiation, the enhancement effect induced by ultrasound almost disappeared, and the cells activity returned to the normal state as control cells without ultrasound stress. The authors stated that the enhancement might be due to defense response of microorganisms evoked by the mechanical stress. That reactions are usually observed when cells are challenged by biotic or abiotic stresses.

Pitt & Ross (2003) used ultrasonic irradiation to increase the growth rate of bacterial cells attached to a polyethylene surface. It was found that low frequency ultrasound (70 kHz) of low intensity (<2 W cm-2) increased the growth rate of the cells compared to growth without ultrasonic waves. They stated that ultrasounds can increase the rate of transport of oxygen and nutrients to the cells and the rate of transport of waste products away from the cells, thus enhancing their growth.

Xie et al. (2009) studied the enhancement effect of low-intensity ultrasound (35 kHz) on anaerobic sludge activity. The experiments showed, that the optimal ultrasonic intensity and irradiation period were 0.2 W cm2 and 10 min, respectively.

To sum up, the optimal ultrasonic intensity and irradiation period are varied in each biological process enhanced by ultrasound and should be find experimentally.
